Arginase I (EC 3.5.3.1; L-arginine amidinohydrolase), is a key mammalian liver enzyme that catalyses, the final step in the urea formation in the Urea cycle, converting arginine into ornithine and urea. Rat liver extract, which has a high content of arginase, was found to have anti-tumour properties in vitro when it was accidentally added to tumour cell culture medium (Burton et al., 1967, Cytolytic action of corticosteroids on thymus and lymphoma cells in vitro. Can J. Biochem. 45, 289-297). Subsequent experiments showed that the anti-tumour properties of the enzyme were due to depletion of arginine, which is an essential amino acid in the culture medium. At below 8 μM levels of arginine, irreparable cell death in cancer cells occurred (Storr & Burton, 1974, The effects of arginine deficiency on lymphoma cells. Br. J. Cancer 30, 50-59).
A more novel aspect of arginine centers on its role as the direct precursor for the synthesis of the potent signalling molecule nitric oxide (NO), which functions as a neurotransmitter, smooth muscle relaxant, and vasodilator. Biosynthesis of NO involves a Ca++, NADPH-dependent reaction catalysed by nitric oxide synthase (NOS). Another recognized role of arginine is that it acts as a precursor, via ornithine, of the polyamines, spermidine and spermine, which participate in diverse physiologic processes including cell proliferation and growth (Wu & Morris, 1998, Arginine metabolism: nitric oxide and beyond. Biochem. J. 336, 1-17).
Arginine also serves as a substrate for several important enzymes, including nitric oxide synthase (NOS). There are three types of NOSs, nNOS, eNOS and iNOS, all convert arginine to nitric oxide and citrulline. The facial flushes induced by NO, for instance, is mediated through nNOS, the neuronal type of NOS. iNOS, the inducible NOS is produced by macrophages and the NO so produced from arginine during septicaemia causes vasodilation in endotoxic shock. eNOS, the endothelial NOS, is produced by endothelial cells in blood vessels. It converts arginine into NO, which then causes de-aggregation of platelets in the endothelial surfaces through cGMP mechanism. NO produced from eNOS in the local endQthelial lining has a half-life of about 5 seconds and diffusion distance of about 2 microns.
The productions of these enzymes are controlled by different NOS genes (NOS1, NOS2, NOS3) encoded in chromosomes 12, 17 & 7, respectively. These genes share strikingly similar genomic structures in size of exons and the location of the splice junctions.
The in vitro anti-tumour activities of arginine depletion were confirmed recently by a group in Scotland, UK (Scott et al., 2000, Single amino acid (arginine) deprivation: rapid and selective death of cultured transformed and malignanat cells. Br. J. Cancer 83, 800-810; Wheatley et al., 2000, Single amino acid (arginine) restriction: Growth and Death of cultured HeLa and Human Diploid Fibroblasts. Cellular Physiol. Biochem. 10, 37-55). Of the 24 different tumour cell lines tested, which included common cancers such as breast, colorectal, lung, prostate and ovaries, all died within 5 days of arginine depletion. Using flow-cytometry studies, the group was able to show that normal cell lines would enter into quiescence for up to several weeks in G0 phase of the cell cycle without any apparent harm. Tumour cells, however, would proceed pass the “R” point in the G1 phase and enter the S phase with deficiency of arginine. Without arginine, which is an irreplaceable amino acid, protein synthesis is deranged. Some cell lines were shown to die from apoptosis. More excitingly, repeated depletions can bringforth tumour kill without “resistance” being developed (Lamb et al., 2000, Single amino acid (arginine) deprivation induces G1 arrest associated with inhibition of Cdk4 expression in cultured human diploid fibroblasts. Experimental Cell Research 225, 238-249).
Despite the promising in vitro data, attempts with arginine depletion to treat cancer in vivo were unsuccessful. The original Storr group attempted to treat tumour-bearing rats with intraperitoneal liver extracts and met with no success (Storr & Burton, 1974, The effects of arginine deficiency on lymphoma cells. Br. J. Cancer 30, 50-59). It is now generally recognized that under normal physiological condition, the blood plasma arginine level and indeed that of other amino acids too, are kept between the normal ranges (100-120 μM) with muscle being the main regulator. In the face of amino acid deficiency, intracellular protein breakdown pathways are activated (proteasomal and lysosomal) releasing amino acids into the circulation (Malumbres & Barbacid, 2001, To cycle or not to cycle: a critical decision in cancer. Nature Reviews, 1, 222-231). This amino acid homeostatic mechanism keeps the various amino acid levels at constant ranges. Thus, previous attempts to deplete arginine with various physical methods or arginine degrading enzymes have failed because of the body's amino acid homeostatic mechanism.
To overcome the problem on the body's natural homeostatic tendencies, Tepic et al. in U.S. Pat. No. 6,261,557 described a therapeutic composition and method for treatment of cancer in which an arginine decomposing enzyme is used in combination with a protein breakdown inhibitors such as insulin in order to prevent the muscles of the body from replenishing the depleted arginine.
Although insulin can act as a protein breakdown inhibitor, it also has far-reaching physiological effects on the human body that may cause fatal problems if blood glucose levels of the patient are not strictly maintained within the narrow normal range. It is therefore an object to the present invention to find improved method of treatment and compositions for the treatment of cancer.